JP2001194298A - Surface plasmon resonance enzyme sensor and method for measuring surface plasmon resonance - Google Patents
Surface plasmon resonance enzyme sensor and method for measuring surface plasmon resonanceInfo
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- JP2001194298A JP2001194298A JP2000312510A JP2000312510A JP2001194298A JP 2001194298 A JP2001194298 A JP 2001194298A JP 2000312510 A JP2000312510 A JP 2000312510A JP 2000312510 A JP2000312510 A JP 2000312510A JP 2001194298 A JP2001194298 A JP 2001194298A
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- Prior art keywords
- surface plasmon
- plasmon resonance
- enzyme
- thin film
- film
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- Investigating Or Analysing Materials By Optical Means (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、酵素を用いて電気
化学的酸化還元作用を起こしながら表面プラズモン共鳴
測定を行うことを特徴とする、表面プラズモン共鳴酵素
センサーおよび表面プラズモン共鳴の測定方法に関する
ものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a surface plasmon resonance enzyme sensor and a method for measuring surface plasmon resonance, wherein a surface plasmon resonance measurement is performed while causing an electrochemical redox action using an enzyme. It is.
【０００２】[0002]
【従来の技術】酵素を用いたバイオセンサーは、血糖値
やコレステロール、尿素やビタミン等多くの生体分子を
選択的に検出する手段として広く研究されている（例え
ば、バイオセンサー、軽部征夫著（共立出版，１９８６
年５月２日出版）を参照）。酸化酵素と電気化学的な検
出方法の組み合わせは中でも特に盛んに研究されてお
り、例えば血糖値センサー及び尿糖値センサーは、幾つ
かのメーカーから商業上利用可能である。2. Description of the Related Art Enzyme-based biosensors have been widely studied as means for selectively detecting many biomolecules such as blood sugar levels, cholesterol, urea, and vitamins (for example, biosensors, written by Masao Karube (Kyoritsu) Publishing, 1986
Published May 2, 2003)). Combinations of oxidases and electrochemical detection methods are among the most actively studied, for example, blood glucose and urinary glucose sensors are commercially available from several manufacturers.
【０００３】酸化酵素を用いたセンサーでは、図３に示
されるように、幾つかの組み合わせが知られており、そ
れらを大別すると、（１） 酵素反応の際の酸素の消費
に伴う、酸素濃度の減少を酸素センサーにより検出する
方法、（２） 酵素反応の生成物である過酸化水素を電
気化学的に酸化して検出する方法、（３） メディエー
ターと呼ばれる酸化還元性の分子と酵素を複合化、ある
いは化学的に結合し、酵素をメディエーターを介して電
極で反応させる方法（酵素消費を伴わない）等が知られ
ている。また、これら（２）と（３）の中間的な方法と
して（４） 酵素反応で生成した過酸化水素をメディエ
ーターと電気的に結合した西洋ワサビペルオキシダーゼ
（ＨＲＰ）を介して還元する方法も報告されている。
（例えば、Vreeke, M., Maiden, R., Heller, A., Ana
l. Chem., 64, 3084-3090(1992)参照）。As shown in FIG. 3, several combinations of sensors using an oxidizing enzyme are known, and they can be roughly classified into (1) oxygen accompanying the consumption of oxygen during the enzymatic reaction. A method of detecting a decrease in concentration by an oxygen sensor, (2) a method of electrochemically oxidizing hydrogen peroxide, which is a product of an enzyme reaction, and (3) a method of detecting a redox molecule called a mediator and an enzyme. Methods of complexing or chemically bonding and reacting an enzyme with an electrode via a mediator (without enzyme consumption) are known. Further, as a method intermediate between these (2) and (3), (4) a method of reducing hydrogen peroxide generated by an enzymatic reaction via horseradish peroxidase (HRP) electrically coupled to a mediator has also been reported. ing.
(Eg, Vreeke, M., Maiden, R., Heller, A., Ana
l. Chem., 64, 3084-3090 (1992)).
【０００４】一方、光を用いたバイオセンサーも研究さ
れている。酸化酵素の生成物を光で検出するためには、
ルミノールのような色素と混合し、化学発光により検出
を行う方法が一般的である。On the other hand, biosensors using light have been studied. To detect oxidase products by light,
A method of mixing with a dye such as luminol and performing detection by chemiluminescence is common.
【０００５】近年、光を用いたバイオセンサーとして表
面プラズモン共鳴（ＳＰＲ）を利用したセンサーが研究
されている。この表面プラズモン共鳴酵素センサー（以
下、ＳＰＲセンサーという）は、金や銀などの基板表面
から数百ｎｍの範囲の屈折率変化を検出できる。実際の
測定では、光を試料の反対面からある角度で入射し、そ
のエバネッセンス波と表面プラズモンとが共鳴する角度
を測定する。In recent years, a sensor using surface plasmon resonance (SPR) has been studied as a biosensor using light. This surface plasmon resonance enzyme sensor (hereinafter, referred to as an SPR sensor) can detect a change in the refractive index in the range of several hundred nm from the surface of a substrate such as gold or silver. In actual measurement, light is incident at a certain angle from the opposite surface of the sample, and the angle at which the evanescent wave and the surface plasmon resonate is measured.
【０００６】測定用光学系の概要を図４（ａ）に示す。
光源から発した光は、くさび形の光に集光し、半円柱状
のプリズム７に入射する。プリズム７の底部には、屈折
率を合わせるための材料（マッチングオイル）を介して
センシング部分６’を配置し、このセンシング部分６’
に全反射の条件下で光を照射する。金属薄膜側に生じる
エバネッセンス波と表面プラズモン波とは、ある入射角
で共鳴を起こす。この時ＣＣＤカメラによって反射光の
強度を測定すると、図４（ｂ）に示すように共鳴が起こ
る角度で、反射率の低い谷が観測される。FIG. 4A shows an outline of a measuring optical system.
The light emitted from the light source is condensed into wedge-shaped light, and enters the semi-cylindrical prism 7. At the bottom of the prism 7, a sensing part 6 'is arranged via a material (matching oil) for adjusting the refractive index, and the sensing part 6'
Is irradiated with light under the condition of total reflection. The evanescent wave and the surface plasmon wave generated on the metal thin film resonate at a certain incident angle. At this time, when the intensity of the reflected light is measured by the CCD camera, a valley having a low reflectance is observed at an angle at which resonance occurs, as shown in FIG.
【０００７】共鳴が起こる角度は、表面の光学的な性質
（屈折率）に依存するため、例えば、抗原−抗体反応に
おいては、センシング部分６’表面に分子が結合する
と、表面の屈折率が変化し、谷のあらわれる角度が僅か
に変化する。この経時変化を測定することにより、表面
の分子の相互作用を高速にモニターすることができる。Since the angle at which resonance occurs depends on the optical properties (refractive index) of the surface, for example, in an antigen-antibody reaction, when a molecule binds to the surface of the sensing portion 6 ', the refractive index of the surface changes. Then, the angle at which the valley appears slightly changes. By measuring the change over time, the interaction of molecules on the surface can be monitored at high speed.
【０００８】ＳＰＲセンサーは、抗原−抗体反応を利用
した免疫センサー、ＤＮＡの検出、及びレセプターとタ
ンパク質の相互作用などの検出に応用されつつある。[0008] The SPR sensor is being applied to an immunosensor utilizing an antigen-antibody reaction, detection of DNA, and detection of interaction between a receptor and a protein.
【０００９】[0009]
【発明が解決しようとする課題】しかしながら、従来の
バイオセンサーのうち、電気化学酵素センサーは、電極
を用いて測定するために、１回の測定に時間と手間がか
かり、大量の検体を迅速に測定することは困難である。
また、光を利用した酵素センサーでは、化学発光や蛍光
を測定する時に試薬を加える必要があるために、操作が
煩雑になり、余分な人手及び／又は高価格の分析装置を
必要とした。また、ＳＰＲセンサーは、センシング部分
に固定された分子認識分子に対して結合した被測定分子
の密度によって屈折率変化を生じさせるものであるた
め、その屈折率変化は分子認識分子の密度以上に高くす
ることができず、測定精度に限界がある。さらに、この
ＳＰＲ測定方法では、測定の間に分子認識分子と被測定
分子とが結合していることが必要であり、分子認識分子
との結合親和性が弱い、或いは結合時間が短い被測定分
子、例えば酵素、過酸化水素のような低分子化合物、ベ
ンゼン置換体、窒素酸化物、神経伝達物質、アミノ酸、
抗原、ＤＮＡオリゴマーなどの分析には適用が困難であ
り、適用範囲が制限される問題があった。However, among the conventional biosensors, the electrochemical enzyme sensor uses an electrode to perform measurement, so that a single measurement takes time and labor, and a large amount of sample can be quickly collected. It is difficult to measure.
In addition, in the case of an enzyme sensor using light, it is necessary to add a reagent when measuring chemiluminescence or fluorescence, so that the operation is complicated, and extra manual labor and / or an expensive analyzer are required. In addition, since the SPR sensor changes the refractive index depending on the density of the molecule to be measured bound to the molecule recognizing molecule fixed to the sensing portion, the change in the refractive index is higher than the density of the molecule recognizing molecule. Measurement, there is a limit to the measurement accuracy. Furthermore, in this SPR measurement method, the molecular recognition molecule and the molecule to be measured need to be bound during the measurement, and the binding affinity with the molecule recognition molecule is weak, or the molecule to be measured has a short binding time. For example, enzymes, low molecular weight compounds such as hydrogen peroxide, benzene substitutes, nitrogen oxides, neurotransmitters, amino acids,
It is difficult to apply to analysis of antigens, DNA oligomers and the like, and there is a problem that the applicable range is limited.
【００１０】この発明は上記のことに鑑み提案されたも
ので、その目的とするところは、測定が簡便であり、か
つ従来法では測定困難であった物質を含む各種の検体を
測定できる表面プラズモン共鳴酵素センサー、および表
面プラズモン共鳴の測定方法を提供することにある。[0010] The present invention has been proposed in view of the above, and it is an object of the present invention to provide a surface plasmon which is easy to measure and can measure various specimens including substances which were difficult to measure by the conventional method. An object of the present invention is to provide a resonance enzyme sensor and a method for measuring surface plasmon resonance.
【００１１】[0011]
【課題を解決するための手段】本発明は、表面プラズモ
ン共鳴装置に用いるセンシング用のセンサーチップにお
いて、光学的に透明な基板上に金または銀の金属薄膜が
形成され、この金属薄膜上は金属薄膜および酵素の両方
と電子移動反応を起こす膜により修飾されていることを
特徴とする表面プラズモン共鳴酵素センサーチップを提
供する。この表面プラズモン共鳴酵素センサーチップに
おいて、電気化学的に酸化還元作用を有する膜は、高分
子であることが好ましい。この電気化学的に酸化還元作
用を有する膜は、膜を介して金属薄膜と電荷のやり取り
をする酵素を含み得る。この電気化学的に酸化還元作用
を有する膜中あるいは膜上、膜と直接あるいは、膜中に
含まれる酵素を介して酸化還元反応を起こす分子を生成
する性質を有する、酵素や細胞、微生物を有し得る。ま
た本発明は、ガラス基板（１）の一方の面側に金薄膜
（２）を形成し、この金薄膜（２）上に作用極（３ａ）
を設けるとともに、酸化還元高分子膜（４）および酵素
固定膜（５）を設けて表面プラズモン共鳴酵素センサー
チップ（６）を構成し、前記ガラス基板（１）の他方の
面側にプリズム（７）を設け、かつ前記金薄膜（２）に
作用極（３ａ）部分となる穴（８ａ）を有するパッキン
（８）を介し液体を保持する空間を有する治具（９）を
押し付け電気化学セル（１０）を構成し、この電気化学
セル（１０）に対極（１１）および参照極（１２）を設
け、各電極をポテンシオスタットに接続し、電気化学的
測定と表面プラズモン共鳴測定とを同時に行うことを特
徴とする表面プラズモン共鳴酵素センサーを提供する。
さらに本発明は、前記表面プラズモン共鳴酵素センサー
を用い、各電極に所定の電位を印加し、電気化学的酸化
還元作用によって生じるセンサーチップ（６）表面上の
物質の電子状態、分子の集合状態、化学的結合状態等の
変化を、プリズム（７）に光を入射し金薄膜（２）側に
生じるエバネッセンス波と表面プラズモンとの共鳴する
角度を測定して検出することを特徴とする表面プラズモ
ン共鳴の測定方法を提供する。According to the present invention, there is provided a sensor chip for sensing used in a surface plasmon resonance apparatus, wherein a metal thin film of gold or silver is formed on an optically transparent substrate, and the metal thin film is formed on the metal thin film. Provided is a surface plasmon resonance enzyme sensor chip characterized by being modified by a film that undergoes an electron transfer reaction with both a thin film and an enzyme. In this surface plasmon resonance enzyme sensor chip, the film having an electrochemical redox effect is preferably a polymer. The electrochemically redox membrane may include an enzyme that exchanges charge with the metal thin film through the membrane. Enzymes, cells, and microorganisms that have the property of generating molecules that cause an oxidation-reduction reaction in or on a membrane that has an electrochemically redox action, directly with the membrane, or via an enzyme contained in the membrane. I can do it. According to the present invention, a gold thin film (2) is formed on one side of a glass substrate (1), and a working electrode (3a) is formed on the gold thin film (2).
And a redox polymer membrane (4) and an enzyme-immobilized membrane (5) are provided to form a surface plasmon resonance enzyme sensor chip (6), and a prism (7) is provided on the other surface of the glass substrate (1). ) And presses a jig (9) having a space for holding a liquid through a packing (8) having a hole (8a) serving as a working electrode (3a) portion on the gold thin film (2). 10), a counter electrode (11) and a reference electrode (12) are provided in the electrochemical cell (10), each electrode is connected to a potentiostat, and an electrochemical measurement and a surface plasmon resonance measurement are simultaneously performed. A surface plasmon resonance enzyme sensor is provided.
Furthermore, the present invention uses the surface plasmon resonance enzyme sensor, applies a predetermined potential to each electrode, and generates an electronic state of a substance on a surface of a sensor chip (6) generated by an electrochemical oxidation-reduction action, an aggregation state of molecules, A surface plasmon resonance characterized in that a change in a chemical bonding state or the like is detected by measuring a resonance angle between an evanescent wave generated on the gold thin film (2) side and surface plasmon by irradiating light to the prism (7). To provide a method for measuring
【００１２】本発明は、光学的に透明な基板と、該基板
上に設けられた金または銀の金属薄膜と、この金属薄膜
に設けられた該金属薄膜及び酵素の両方と電子移動反応
を起こす膜とを有するセンシング部分を含むことを特徴
とする表面プラズモン共鳴酵素センサーを提供する。こ
の表面プラズモン共鳴酵素センサーにおいて、前記電子
移動を起こす膜は、高分子膜として良い。この表面プラ
ズモン共鳴酵素センサーにおいて、前記電子移動反応を
起こす膜は、該膜を介して前記金属薄膜と電荷のやり取
りをする酵素を含み得る。この表面プラズモン共鳴酵素
センサーにおいて、前記電子移動反応を起こす膜と直
接、該膜中に含まれる酵素を介して、またはその両方の
中から選択される１つと酸化還元反応を起こす分子を生
成する性質を有する、酵素、細胞、微生物からなる群か
ら選択される１つを含み得る。本発明はまた、ガラス基
板（１）と、該ガラス基板（１）の一方の面側に設けら
れた金薄膜（２）と、該金薄膜（２）上に設けられた作
用極（３ａ）、酸化還元高分子膜（４）及び酵素固定膜
（５）とを含むセンシング部分（６）と、前記ガラス基
板（１）の他方の面側に設けられたプリズム（７）と、
前記金薄膜（２）に接触して設けられ、前記作用極（３
ａ）部分となる穴（８ａ）を有するパッキン（８）を介
して電解液を保持する空間を有する治具（９）とを有す
る電気化学セル（１０）と；該電気化学セル（１０）内
に保持される電解液に接して離間配置された対極（１
１）および参照極（１２）とを含み；これらの作用極
（３ａ）、対極（１１）および参照極（１２）をポテン
シオスタットに接続して電気化学的測定と表面プラズモ
ン共鳴測定とを同時に行うように構成されたことを特徴
とする表面プラズモン共鳴酵素センサーを提供する。さ
らに本発明は、前記の表面プラズモン共鳴酵素センサー
を用い、各電極に所定の電位を印加し、電気化学的酸化
還元作用によって生じるセンシング部分（６）表面上の
物質の電子状態、分子の集合状態、化学的結合状態およ
び物質移動からなる少なくとも１つの変化を、プリズム
（７）に光を入射し金薄膜（２）側に生じるエバネッセ
ンス波と表面プラズモンとの共鳴する角度を測定して検
出する工程を含むことを特徴とする表面プラズモン共鳴
の測定方法を提供する。According to the present invention, an electron transfer reaction occurs between an optically transparent substrate, a gold or silver metal thin film provided on the substrate, and both the metal thin film and an enzyme provided on the metal thin film. A surface plasmon resonance enzyme sensor comprising a sensing portion having a membrane. In this surface plasmon resonance enzyme sensor, the film that causes the electron transfer may be a polymer film. In this surface plasmon resonance enzyme sensor, the film that causes the electron transfer reaction may include an enzyme that exchanges charge with the metal thin film via the film. In this surface plasmon resonance enzyme sensor, the property of generating a molecule that causes an oxidation-reduction reaction with one selected from the membrane that causes the electron transfer reaction, directly through an enzyme contained in the membrane, or both. And one selected from the group consisting of enzymes, cells, and microorganisms. The present invention also provides a glass substrate (1), a gold thin film (2) provided on one side of the glass substrate (1), and a working electrode (3a) provided on the gold thin film (2). A sensing part (6) including a redox polymer membrane (4) and an enzyme-immobilized membrane (5); a prism (7) provided on the other surface side of the glass substrate (1);
The working electrode (3) is provided in contact with the gold thin film (2).
a) a jig (9) having a space for holding an electrolyte through a packing (8) having a hole (8a) to be a part; and an electrochemical cell (10); Counter electrode (1
1) and a reference electrode (12); the working electrode (3a), the counter electrode (11) and the reference electrode (12) are connected to a potentiostat to perform electrochemical measurement and surface plasmon resonance measurement simultaneously. The present invention provides a surface plasmon resonance enzyme sensor characterized in that it is configured to perform the same. Further, the present invention uses the above-mentioned surface plasmon resonance enzyme sensor, applies a predetermined potential to each electrode, and generates a sensing portion (6) generated by electrochemical redox action. Detecting a change in at least one of a chemical bonding state and mass transfer by irradiating the prism (7) with light and measuring an angle of resonance between an evanescent wave generated on the side of the gold thin film (2) and the surface plasmon. And a method for measuring surface plasmon resonance.
【００１３】本発明は、光学的透明体と、該透明体上の
少なくとも一部に設けられた表面プラズモン共鳴を起こ
し得る物質を含む薄膜とを有するセンシング部を含み、
該薄膜は、該薄膜または該薄膜上に設けられた導体と電
気化学反応する物質と、酵素との両方により修飾されて
いることを特徴とする表面プラズモン共鳴酵素センサー
を提供する。この表面プラズモン共鳴酵素センサーにお
いて、前記薄膜は高分子とし得る。或いは、薄膜は、金
または銀の薄膜とし得る。この表面プラズモン共鳴酵素
センサーにおいて、前記薄膜は、該薄膜を介して電荷の
やり取りをする酵素を含み得る。この表面プラズモン共
鳴酵素センサーにおいて、前記薄膜と直接、該膜中に含
まれる酵素を介して、またはその両方の中から選択され
る１つと酸化還元反応を起こす分子を生成する性質を有
する、酵素、細胞および微生物からなる群から選択され
る１つを有して良い。また本発明は、ガラス基体（１）
と、該ガラス基体（１）の一方の面側に設けられた金薄
膜（２）と、該金薄膜（２）上に設けられた作用極（３
ａ）、酸化還元高分子膜（４）及び酵素固定膜（５）と
を含むセンシング部分（６）と、前記金薄膜（２）に接
触して設けられ、前記作用極（３ａ）部分となる穴（８
ａ）を有するパッキン（８）を介して電解液を保持する
空間を有する治具（９）とを有する電気化学セル（１
０）と；該電気化学セル（１０）内に保持される電解液
に接して離間配置された対極（１１）および参照極（１
２）とを含み；これらの作用極（３ａ）、対極（１１）
および参照極（１２）を用いて電気化学的測定と表面プ
ラズモン共鳴測定とを同時に行うように構成されたこと
を特徴とする表面プラズモン共鳴酸素センサーを提供す
る。さらに本発明は、前記表面プラズモン共鳴酵素セン
サーを用い、各電極に酸化還元高分子膜（４）または酵
素固定膜（５）に電気化学的反応を起こさせる電位を印
加、または時間的に変化する電位を印加し、電気化学的
酸化還元作用によって生じるセンシング部分（６）の物
質の電子状態、分子の集合状態、化学的結合状態及び物
質移動からなる群から選択される少なくとも１つの変化
を、金薄膜（２）と反対側のガラス基体（１）側から光
を入射し、金薄膜（２）で起こる表面プラズモン共鳴現
象に基づいて検出する工程を含むことを特徴とする表面
プラズモン共鳴の測定方法を提供する。The present invention includes a sensing unit having an optically transparent body and a thin film provided on at least a part of the transparent body and containing a substance capable of causing surface plasmon resonance,
The surface plasmon resonance enzyme sensor is characterized in that the thin film is modified by both an enzyme and a substance that electrochemically reacts with the thin film or a conductor provided on the thin film. In this surface plasmon resonance enzyme sensor, the thin film may be a polymer. Alternatively, the film may be a gold or silver film. In the surface plasmon resonance enzyme sensor, the thin film may include an enzyme that exchanges electric charges through the thin film. In the surface plasmon resonance enzyme sensor, an enzyme having a property of generating a molecule that causes an oxidation-reduction reaction with one selected from the thin film directly, via an enzyme contained in the film, or both, It may have one selected from the group consisting of cells and microorganisms. The present invention also provides a glass substrate (1)
A gold thin film (2) provided on one side of the glass substrate (1); and a working electrode (3) provided on the gold thin film (2).
a), a sensing portion (6) including a redox polymer film (4) and an enzyme immobilized film (5), and a sensing portion (6) provided in contact with the gold thin film (2) to serve as the working electrode (3a) portion. Hole (8
a jig (9) having a space for holding an electrolyte through a packing (8) having a) (a).
0) and; a counter electrode (11) and a reference electrode (1) which are separately arranged in contact with the electrolytic solution held in the electrochemical cell (10).
2) and these working electrodes (3a) and counter electrodes (11).
And a surface plasmon resonance oxygen sensor characterized in that electrochemical measurement and surface plasmon resonance measurement are simultaneously performed using the reference electrode (12). Further, in the present invention, using the surface plasmon resonance enzyme sensor, a potential for causing an electrochemical reaction on the redox polymer film (4) or the enzyme immobilized film (5) is applied to each electrode, or changes with time. A potential is applied, and at least one change selected from the group consisting of the electronic state of the substance of the sensing portion (6), the aggregate state of molecules, the chemical bond state, and the mass transfer caused by the electrochemical redox action is determined by gold. A method for measuring surface plasmon resonance, comprising the step of irradiating light from the glass substrate (1) side opposite to the thin film (2) and detecting based on a surface plasmon resonance phenomenon occurring in the gold thin film (2). I will provide a.
【００１４】[0014]
【発明の実施の形態】本発明者らは、光を利用し、かつ
蛍光や発光試薬を加えることなく酵素を利用したバイオ
センサーを実現するための検討を行ったところ、酵素反
応生成物である過酸化水素が西洋ワサビペルオキシダー
ゼ（ＨＲＰ）を介して酸化還元高分子膜を酸化させると
ＳＰＲセンサーに対して高い感度を示すことを見出し
た。また、一度酸化した酸化還元高分子膜は、電気化学
的に還元状態に容易に戻すことができ、同時にＳＰＲの
共鳴角も元の位置に戻ることを見出し、本発明に至っ
た。BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have studied to realize a biosensor using an enzyme without using a fluorescent or luminescent reagent by using light, and found that an enzyme reaction product is obtained. It has been found that when hydrogen peroxide oxidizes a redox polymer membrane via horseradish peroxidase (HRP), it exhibits high sensitivity to an SPR sensor. Further, the present inventors have found that the redox polymer film once oxidized can be easily returned electrochemically to the reduced state, and at the same time, the resonance angle of SPR also returns to the original position, and thus the present invention has been achieved.
【００１５】以下に、図面を参照して本発明を実施例に
より詳細に説明する。なお、本発明は以下の実施例のみ
に限定されるものではない。Hereinafter, the present invention will be described in more detail with reference to the drawings. The present invention is not limited only to the following examples.
【００１６】[0016]
【実施例】実施例１ 本発明の一実施例を図面を参照して説明する。図１
（ａ）は、本発明の表面プラズモン共鳴酵素センサーの
センシング部分６を説明するための概略図であり、
（ｂ）はこの表面プラズモン共鳴酵素センサーによる反
応の概略図である。図１（ａ）において、１は光学的に
透明な基板、例えばＢＫ７からなるガラス基板である。
２はその上面に形成された金又は銀の金属薄膜であり、
本実施例では金薄膜を用いている。３はこの金薄膜２上
に設けられた作用極３ａのリード線、４は、金薄膜２上
に設けられた西洋ワサビペルオキシダーゼ（ＨＲＰ）を
含む酸化還元高分子膜、５はさらにその上に設けられた
酵素固定膜であり、これらによって表面プラズモン共鳴
酵素センサーのセンシング部分（センサーチップ）６が
構成されている。Embodiment 1 An embodiment of the present invention will be described with reference to the drawings. FIG.
(A) is a schematic diagram for explaining the sensing part 6 of the surface plasmon resonance enzyme sensor of the present invention,
(B) is a schematic diagram of the reaction by this surface plasmon resonance enzyme sensor. In FIG. 1A, reference numeral 1 denotes an optically transparent substrate, for example, a glass substrate made of BK7.
2 is a gold or silver metal thin film formed on the upper surface,
In this embodiment, a gold thin film is used. 3 is a lead wire of a working electrode 3a provided on the gold thin film 2, 4 is a redox polymer film containing horseradish peroxidase (HRP) provided on the gold thin film 2, and 5 is further provided thereon. And a sensing part (sensor chip) 6 of the surface plasmon resonance enzyme sensor.
【００１７】図２は、酵素膜を有する表面プラズモン共
鳴酵素センサーを用いた電気化学反応と組み合わせて酵
素の性質を検出する電気化学セルを示す図である。FIG. 2 shows an electrochemical cell for detecting the properties of an enzyme in combination with an electrochemical reaction using a surface plasmon resonance enzyme sensor having an enzyme membrane.
【００１８】この電気化学セル１０は次のようにして作
製される。ガラス基板１，例えば松浪硝子工業社製の厚
さ０．３ｍｍのガラスを用いたガラス基板１の一方の面
側に、金薄膜２を形成する。この実施例においては、日
本シード研究所社製の装置を用い、スパッタ法によって
厚さ５０ｎｍの金薄膜２を形成した。この金薄膜２上に
西洋ワサビペルオキシダーゼ（ＨＲＰ）を含む酸化還元
高分子（オスミウムポリマー、ＢＡＳ社製）を塗布し過
酸化水素を検出するバイオセンサー電極を作製した。酸
化還元高分子膜４は、電極と酵素の両方と電気移動反応
を起こすものであればよく、オスミウム化合物の他にも
フェロセン化合物、有機酸化還元試薬、金属錯体、酸化
還元酵素など、バイオセンサーにおいて一般的にメディ
エーターとなる物質が利用できる。The electrochemical cell 10 is manufactured as follows. A gold thin film 2 is formed on one side of a glass substrate 1, for example, a glass substrate 1 made of glass having a thickness of 0.3 mm manufactured by Matsunami Glass Industry Co., Ltd. In this example, a gold thin film 2 having a thickness of 50 nm was formed by a sputtering method using an apparatus manufactured by Japan Seed Research Institute. A redox polymer (osmium polymer, manufactured by BAS) containing horseradish peroxidase (HRP) was applied on the gold thin film 2 to prepare a biosensor electrode for detecting hydrogen peroxide. The redox polymer film 4 only needs to cause an electrotransfer reaction with both the electrode and the enzyme. In addition to the osmium compound, a ferrocene compound, an organic redox reagent, a metal complex, an oxidoreductase, etc. Generally, a substance serving as a mediator can be used.
【００１９】また、電気化学的に酸化還元作用を有する
酸化還元高分子膜４は、この膜と直接、該膜中に含まれ
る酵素を介して、またはその両方の中から選択される１
つと酸化還元反応を起こす分子を生成する性質を有す
る。上記酵素としては、従来より周知の酵素を用いて良
く、例えば、酵素ハンドブック（朝倉書店１９８２年）
の「１．酸化還元酵素」中に記載された各種の酵素また
は、Enzyme Handbook 10, Class 1.1.1.150-1.1.99.26
Oxidoreductases (Eds.: D. Schomburg, D. Stephan, S
pringer-Verlag 1995)に記載された酵素、好ましくは、
ペルオキシダーゼ、グルコースオキシダーゼ、グルタミ
ン酸化酵素、ヒスタミン酸化酵素、コリン酸化酵素、コ
レステロール酸化酵素を挙げることができる。酵素の代
替として、これらの酵素を生産する細胞または微生物
を、十分に酵素の単離精製を行うことなしに、或いは未
精製のままで用いることもできる。この目的で用いられ
る細胞としては、動物の脳細胞、植物の組織細胞など、
好ましくは、上記酵素を生産する細胞を挙げることがで
きる。また前記微生物としては、大腸菌、枯草菌、及び
藻類など、好ましくは、上記酵素を合成可能な遺伝子組
換え大腸菌を挙げることができる。さらに、酵素の代替
として、モレキュラーインプリント法やカタリティック
アンチボディー法で合成された分子を用いることもでき
る。The redox polymer membrane 4 having an electrochemical oxidation-reduction action can be selected from the membrane directly, via an enzyme contained in the membrane, or both.
They have the property of generating molecules that cause a redox reaction. As the enzyme, a conventionally well-known enzyme may be used. For example, an enzyme handbook (Asakura Shoten, 1982)
Various enzymes described in “1. Redox enzymes” of Enzyme Handbook 10, Class 1.1.1.150-1.1.99.26
Oxidoreductases (Eds .: D. Schomburg, D. Stephan, S
pringer-Verlag 1995), preferably,
Examples include peroxidase, glucose oxidase, glutamine oxidase, histamine oxidase, choline oxidase, and cholesterol oxidase. As an alternative to enzymes, cells or microorganisms producing these enzymes can be used without sufficient isolation and purification of the enzymes or without purification. Cells used for this purpose include animal brain cells, plant tissue cells, and the like.
Preferably, a cell that produces the above enzyme can be mentioned. Examples of the microorganism include Escherichia coli, Bacillus subtilis, and algae, and preferably, genetically modified Escherichia coli capable of synthesizing the above enzyme. Further, as an alternative to the enzyme, a molecule synthesized by a molecular imprinting method or a catalytic antibody method can be used.
【００２０】電極を構成するガラス基板１の他方のガラ
ス面にＢＫ７と同一の屈折率を持つ液体（マッチングオ
イル）を塗布し、電気化学工業社製ＳＰＲ測定装置ＳＰ
Ｒ−２０のプリズム７に密着させた。この電極の酵素固
定膜５面に円形の穴８ａのあるシリコーンラバーのパッ
キン８を介し電解液を保持する空間を有する治具９を押
し付け、電気化学セル１０を構成した。金薄膜２にリー
ド線３を取り付け、パッキン８の穴８ａの部分を作用極
３ａとし、また、この電気化学セル１０に対極１１とな
る白金線と参照極１２となる塩橋付きの銀塩化銀電極を
組み込み、セル１０内に電解液を入れてＳＰＲ測定装置
を構成した。電解液としては、リン酸、カコジル酸、過
塩素酸、六フッ化リン酸、四フッ化ホウ酸、有機酸のそ
れぞれの金属塩、アンモニウム塩、トリス(tris(hydrox
ymethyl)aminomethane)塩の水溶液などが、特に好まし
くはリン酸緩衝液、トリス緩衝液が用いられる。A liquid (matching oil) having the same refractive index as BK7 is applied to the other glass surface of the glass substrate 1 constituting the electrode, and an SPR measuring device SP manufactured by Denki Kagaku Kogyo KK
It was closely attached to the prism 7 of R-20. A jig 9 having a space for holding an electrolytic solution was pressed through a silicone rubber packing 8 having a circular hole 8a on the surface of the enzyme-immobilized membrane 5 of the electrode to form an electrochemical cell 10. The lead wire 3 is attached to the gold thin film 2, the hole 8 a of the packing 8 is used as a working electrode 3 a, and a platinum wire serving as a counter electrode 11 and a silver-silver chloride with a salt bridge serving as a reference electrode 12 are provided in the electrochemical cell 10. The electrode was assembled, and an electrolytic solution was put in the cell 10 to constitute an SPR measuring device. Examples of the electrolyte include phosphoric acid, cacodylic acid, perchloric acid, hexafluorophosphoric acid, tetrafluoroboric acid, metal salts of organic acids, ammonium salts, and tris (hydrox
An aqueous solution of ymethyl) aminomethane) salt or the like is particularly preferably used, and a phosphate buffer and a Tris buffer are used.
【００２１】電気化学セル１０内にリン酸緩衝液を満た
し、扶桑製作所製のポテンシオスタットに作用極リード
線３、対極１１、参照極１２、をそれぞれ接続し、ＨＲ
Ｐの酵素活性が現れる電位を作用極３ａに印加し、電気
化学測定を行った。同時にＳＰＲ−２０によって、図４
（ａ）、（ｂ）に示したように、ＳＰＲ測定を行った。
この装置でまずＨＲＰの基質を含まない条件でサイクリ
ックボルタメトレー法（以下、ＣＶという）で測定を行
ったところ、電気化学的電流は得られず、ＳＰＲ測定で
電極電位に依存した信号は得られなかった。The electrochemical cell 10 is filled with a phosphate buffer, and the working electrode lead wire 3, the counter electrode 11, and the reference electrode 12 are connected to a potentiostat manufactured by Fuso Seisakusho, respectively.
A potential at which the enzyme activity of P appeared was applied to the working electrode 3a, and an electrochemical measurement was performed. At the same time, by SPR-20, FIG.
SPR measurement was performed as shown in (a) and (b).
When a cyclic voltammetry method (hereinafter referred to as “CV”) was first performed using this apparatus under conditions that did not include an HRP substrate, no electrochemical current was obtained, and a signal depending on the electrode potential in SPR measurement was not obtained. Could not be obtained.
【００２２】次に、ＨＲＰの基質である過酸化水素を含
む条件でＣＶを行ったところ、酵素反応に特徴的なシグ
モイド形の曲線を得た。また、ＳＰＲ測定ではＣＶと同
じ電位でシグナルの変化が見られ、上記の構造を持つ表
面プラズモン共鳴酵素センサーのセンシング部分６のＳ
ＰＲ測定で電気化学的酵素活性の発現状態、すなわちセ
ンシング部分６表面上の物質の電子状態、分子の集合状
態、化学的結合状態及び物質移動の変化を測定すること
ができた。Next, when CV was performed under conditions including hydrogen peroxide which is a substrate of HRP, a sigmoidal curve characteristic of the enzyme reaction was obtained. In the SPR measurement, a signal change was observed at the same potential as the CV, and the SPR of the sensing portion 6 of the surface plasmon resonance enzyme sensor having the above-described structure was observed.
By the PR measurement, the expression state of the electrochemical enzyme activity, that is, the change of the electronic state of the substance on the surface of the sensing portion 6, the state of molecular assembly, the chemical bonding state, and the mass transfer could be measured.
【００２３】実施例２ 実施例１と同様の方法によってＨＲＰを含む電気化学反
応型バイオセンサーを作り、電気化学ＳＰＲ測定装置を
構成した。作用極３ａである金薄膜２にＨＲＰの酵素活
性が現れる電位を印加し、ＨＲＰの基質である過酸化水
素濃度を変化させ、作用極３ａに流れる電流とＳＰＲ信
号を測定した。Example 2 An electrochemical reaction type biosensor containing HRP was produced in the same manner as in Example 1, and an electrochemical SPR measuring apparatus was constructed. The potential at which the enzyme activity of HRP appears was applied to the gold thin film 2 as the working electrode 3a, the concentration of hydrogen peroxide as the substrate of HRP was changed, and the current flowing through the working electrode 3a and the SPR signal were measured.
【００２４】その結果、過酸化水素濃度が低いときは電
流は濃度に比例し、センサーとして働くことが分かっ
た。また、ＳＰＲ信号も濃度に比例し、ＳＰＲ測定によ
っても、定量測定が可能であることがわかった。さら
に、電気化学セル１０を基質を含まない溶液に戻すとセ
ンサーの応答は最初の状態に戻り、繰り返し測定が簡便
に行えることがわかった。As a result, it was found that when the concentration of hydrogen peroxide was low, the current was proportional to the concentration and worked as a sensor. Also, the SPR signal was proportional to the concentration, and it was found that quantitative measurement was possible by SPR measurement. Furthermore, when the electrochemical cell 10 was returned to a solution containing no substrate, the response of the sensor returned to the initial state, and it was found that repeated measurements could be easily performed.
【００２５】次に、作用極電位をＨＲＰの酵素活性が生
じない電位に保持した場合、過酸化水素の濃度に対する
センサーの応答は電流によっても、また、ＳＰＲ測定に
よっても得られなかった。Next, when the working electrode potential was maintained at a potential at which the enzymatic activity of HRP did not occur, the response of the sensor to the concentration of hydrogen peroxide was not obtained by the current nor by the SPR measurement.
【００２６】したがって、このセンサーのＳＰＲ測定で
得られる過酸化水素に対する応答は、過酸化水素の濃度
が変化したために金薄膜表面での屈折率が変化したのを
検出したのではなく、酵素を含む酸化還元高分子膜４が
電気化学作用によって活性化された酵素反応の影響によ
って屈折率の変化を起こし、これをＳＰＲ法によって検
出したものであることがわかる。Therefore, the response to hydrogen peroxide obtained by the SPR measurement of this sensor does not indicate that the refractive index on the surface of the gold thin film has changed due to the change in the concentration of hydrogen peroxide, but includes the enzyme. It can be seen that the redox polymer film 4 caused a change in the refractive index due to the effect of the enzyme reaction activated by the electrochemical action, and this was detected by the SPR method.
【００２７】実施例３ 実施例１と同様な方法で表面プラズモン共鳴酵素センサ
ーのセンシング部分６を作製し、ＨＲＰの基質のない溶
液中でＨＲＰが活性な状態になるように電極電位を設定
した。このとき、電極に電流は流れず、ＳＰＲ測定装置
の応答は一定値を示した。この状態に基質を加え、電流
が流れない状態での電極電位（静止電位）の変化を測定
したところ、時間と共にＨＲＰが基質を消費し酸化還元
反応を起こす酸化還元高分子膜４の酸化型と還元型の濃
度比の変化に由来する電位変化が測定された。Example 3 A sensing portion 6 of a surface plasmon resonance enzyme sensor was prepared in the same manner as in Example 1, and the electrode potential was set so that HRP was activated in a solution containing no HRP substrate. At this time, no current flowed through the electrodes, and the response of the SPR measurement device showed a constant value. When a substrate was added to this state and the change in electrode potential (resting potential) in a state where no current flowed was measured, the oxidized form of the redox polymer film 4 in which HRP consumed the substrate and caused a redox reaction with time was measured. The potential change resulting from the change in the concentration ratio of the reduced form was measured.
【００２８】同時に行ったＳＰＲ測定では、この酸化型
と還元型の濃度比を反映する屈折率の変化が測定され
た。この場合、測定時にはポテンシオスタットは必要な
く、より簡便な方法で酸化還元作用を有する高分子膜を
用いた表面プラズモン共鳴酵素センサーを構築すること
ができた。In the SPR measurement performed at the same time, a change in the refractive index reflecting the concentration ratio between the oxidized form and the reduced form was measured. In this case, a potentiostat was not required for measurement, and a surface plasmon resonance enzyme sensor using a polymer film having an oxidation-reduction action could be constructed by a simpler method.
【００２９】実施例４ まず、実施例１と同様にＨＲＰを含む酸化還元高分子を
塗布した表面プラズモン共鳴酵素センサーのセンシング
部６を作製した。この酸化還元高分子膜４上に牛アルブ
ミン（シグマ社製）を含むグルコース酸化酵素（ＧＯ
Ｄ）溶液を塗布し、このセンシング部分６をグルタルア
ルデヒド（シグマ社製）蒸気に曝してＧＯＤ固定化膜層
５を形成した。Example 4 First, as in Example 1, a sensing unit 6 of a surface plasmon resonance enzyme sensor coated with a redox polymer containing HRP was prepared. Glucose oxidase (GO) containing bovine albumin (Sigma) on the redox polymer film 4
D) A solution was applied, and this sensing part 6 was exposed to glutaraldehyde (Sigma) vapor to form a GOD-immobilized film layer 5.
【００３０】この表面プラズモン共鳴酵素センサーのセ
ンシング部分６を実施例２と同様な方法でＨＲＰの酵素
活性が得られる電位をそれぞれの電極に印加して、セン
サーのグルコースに対する応答を調べた。The sensing part 6 of this surface plasmon resonance enzyme sensor was applied to each electrode with a potential at which the enzyme activity of HRP was obtained in the same manner as in Example 2, and the response of the sensor to glucose was examined.
【００３１】その結果、グルコースの濃度に応じて電流
が増加し、ＳＰＲ測定でもこれに相当する結果が得られ
た。また、ＳＰＲ測定での検出限界は１０-5Ｍ以下であ
り、実用的に十分な感度が得られた。As a result, the current increased in accordance with the concentration of glucose, and a result corresponding to this was obtained in SPR measurement. In addition, the detection limit in SPR measurement was 10 −5 M or less, and sufficient sensitivity for practical use was obtained.
【００３２】この実施例と同様の応答は、他の酸化酵素
である、グルタミン酸化酵素、ヒスタミン酸化酵素（Y-
H. Choi, R. Matsuzaki, T. Fukui, E. Shimizu, T. Yo
rifuji, H. Sato, Y. Ozaki and K. Tanizawa, J. Bio
l. Chem., 270, 4712-20 (1995)）、コリン酸化酵素、
コレステロール酸化酵素膜を用いたときにも観測され、
ＳＰＲ法を利用した幅広い酵素センサーを構築できるこ
とがわかった。The response similar to that of this example is obtained by using other oxidases such as glutamine oxidase and histamine oxidase (Y-
H. Choi, R. Matsuzaki, T. Fukui, E. Shimizu, T. Yo
rifuji, H. Sato, Y. Ozaki and K. Tanizawa, J. Bio
l. Chem., 270, 4712-20 (1995)), choline oxidase,
Also observed when using cholesterol oxidase membrane,
It was found that a wide range of enzyme sensors using the SPR method could be constructed.
【００３３】[0033]
【発明の効果】本発明によれば、酵素を用いて電気化学
的酸化還元作用を起こしながら表面プラズモン共鳴測定
を行うことによって、１回の測定を短時間で行うことが
でき、大量の検体を迅速に測定することが可能となる。
また、本発明によれば、測定操作が簡略になり、人手を
省き、かつ安価な分析装置を構成することができる。さ
らに本発明によれば、センシング部分に被測定分子を捕
獲せずに測定することが可能となり、従来のＳＰＲ法で
は検出が困難であった分子認識分子との結合親和性が弱
い、或いは結合時間が短い被測定分子、例えば酵素、過
酸化水素のような低分子化合物を高感度で検出すること
ができる。従って、本発明によれば、被測定分子の種類
のバリエーションを広げることができる。さらにまた、
本発明によれば、センシング部分に被測定分子を捕獲せ
ずに測定することができるので、最終的な感度が被測定
物の密度と無関係となるので、測定精度を大幅に向上す
ることができる。According to the present invention, a single measurement can be performed in a short time by performing a surface plasmon resonance measurement while causing an electrochemical oxidation-reduction action using an enzyme, and a large amount of a sample can be obtained. It becomes possible to measure quickly.
Further, according to the present invention, the measurement operation can be simplified, manpower can be saved, and an inexpensive analyzer can be configured. Further, according to the present invention, measurement can be performed without capturing the molecule to be measured in the sensing portion, and the binding affinity with the molecule recognition molecule, which was difficult to detect by the conventional SPR method, is low, or the binding time is low. Can be detected with high sensitivity, for example, a low-molecular-weight compound such as an enzyme or hydrogen peroxide, which has a short measurement time. Therefore, according to the present invention, it is possible to widen the variation of the types of the molecules to be measured. Furthermore,
According to the present invention, the measurement can be performed without capturing the molecule to be measured in the sensing portion, so that the final sensitivity is independent of the density of the object to be measured, so that the measurement accuracy can be greatly improved. .
【図１】 （ａ）は、本発明の一実施例にかかる表面プ
ラズモン共鳴酵素センサーのセンシング部分の概略図で
ある。（ｂ）は、反応の概略を示す図である。FIG. 1A is a schematic diagram of a sensing part of a surface plasmon resonance enzyme sensor according to one embodiment of the present invention. (B) is a figure which shows the outline of a reaction.
【図２】 酵素膜を有するＳＰＲセンサーを用いた電気
化学反応と組み合わせて酵素の基質を検出する電気化学
セルを示す。FIG. 2 shows an electrochemical cell for detecting an enzyme substrate in combination with an electrochemical reaction using an SPR sensor having an enzyme membrane.
【図３】 各種の測定方法を説明する概略図である。FIG. 3 is a schematic diagram illustrating various measurement methods.
【図４】 （ａ）は、ＳＰＲ測定の光学系を示す概略図
である。（ｂ）はＣＣＤカメラでの反射光の強度を測定
した状態を示す概略図である。FIG. 4A is a schematic diagram showing an optical system for SPR measurement. (B) is a schematic diagram showing a state where the intensity of the reflected light is measured by the CCD camera.
１ ガラス基板 ２ 金薄膜 ３ リード線 ３ａ 作用極 ４ 酸化還元高分子膜 ５ 酵素固定膜 ６ 表面プラズモン共鳴酵素センサーのセンシング部
分（センサーチップ） ７ プリズム ８ パッキン ８ａ 穴 ９ 治具 １０ 電気化学セル １１ 対極 １２ 参照極DESCRIPTION OF SYMBOLS 1 Glass substrate 2 Gold thin film 3 Lead wire 3a Working electrode 4 Redox polymer film 5 Enzyme fixed film 6 Sensing part of surface plasmon resonance enzyme sensor (sensor chip) 7 Prism 8 Packing 8a Hole 9 Jig 10 Electrochemical cell 11 Counter electrode 12 Reference pole
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｃ１２Ｑ 1/28 Ｃ１２Ｑ 1/28 Ｇ０１Ｎ 27/327 Ｇ０１Ｎ 33/543 ５９５ 33/543 ５９５ 27/30 ３５１ ──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) C12Q 1/28 C12Q 1/28 G01N 27/327 G01N 33/543 595 33/543 595 27/30 351
Claims (19)
ング用のセンサーチップにおいて、光学的に透明な基板
上に金または銀の金属薄膜が形成され、この金属薄膜上
は金属薄膜および酵素の両方と電子移動反応を起こす膜
により修飾されていることを特徴とする表面プラズモン
共鳴酵素センサーチップ。In a sensor chip for sensing used in a surface plasmon resonance apparatus, a gold or silver metal thin film is formed on an optically transparent substrate, and on this metal thin film, both a metal thin film and an enzyme are transferred with electrons. A surface plasmon resonance enzyme sensor chip characterized by being modified by a membrane that causes a reaction.
高分子であることを特徴とする請求項１に記載の表面プ
ラズモン共鳴酵素センサーチップ。2. The surface plasmon resonance enzyme sensor chip according to claim 1, wherein the membrane having an electrochemical oxidation-reduction action is a polymer.
が、膜を介して金属薄膜と電荷のやり取りをする酵素を
含むことを特徴とする請求項１に記載の表面プラズモン
共鳴酵素センサーチップ。3. The surface plasmon resonance enzyme sensor chip according to claim 1, wherein the film having an electrochemical redox action includes an enzyme that exchanges electric charge with a metal thin film via the film.
あるいは膜上、膜と直接あるいは、膜中に含まれる酵素
を介して酸化還元反応を起こす分子を生成する性質を有
する、酵素や細胞、微生物を有することを特徴とする請
求項１に記載の表面プラズモン共鳴酵素センサーチッ
プ。4. An enzyme or cell having the property of generating a molecule that causes an oxidation-reduction reaction in or on a membrane having an electrochemical redox action, directly with the membrane, or via an enzyme contained in the membrane. The surface plasmon resonance enzyme sensor chip according to claim 1, further comprising a microorganism.
（２）を形成し、この金薄膜（２）上に作用極（３ａ）
を設けるとともに、酸化還元高分子膜（４）および酵素
固定膜（５）を設けて表面プラズモン共鳴酵素センサー
チップ（６）を構成し、前記ガラス基板（１）の他方の
面側にプリズム（７）を設け、かつ前記金薄膜（２）に
作用極（３ａ）部分となる穴（８ａ）を有するパッキン
（８）を介し液体を保持する空間を有する治具（９）を
押し付け電気化学セル（１０）を構成し、この電気化学
セル（１０）に対極（１１）および参照極（１２）を設
け、各電極をポテンシオスタットに接続し、電気化学的
測定と表面プラズモン共鳴測定とを同時に行うことを特
徴とする表面プラズモン共鳴酵素センサー。5. A thin gold film (2) is formed on one side of a glass substrate (1), and a working electrode (3a) is formed on the thin gold film (2).
And a redox polymer membrane (4) and an enzyme-immobilized membrane (5) are provided to form a surface plasmon resonance enzyme sensor chip (6), and a prism (7) is provided on the other surface of the glass substrate (1). ) And presses a jig (9) having a space for holding a liquid through a packing (8) having a hole (8a) serving as a working electrode (3a) portion on the gold thin film (2). 10), a counter electrode (11) and a reference electrode (12) are provided in the electrochemical cell (10), each electrode is connected to a potentiostat, and an electrochemical measurement and a surface plasmon resonance measurement are simultaneously performed. A surface plasmon resonance enzyme sensor.
センサーを用い、各電極に所定の電位を印加し、電気化
学的酸化還元作用によって生じるセンサーチップ（６）
表面上の物質の電子状態、分子の集合状態、化学的結合
状態等の変化を、プリズム（７）に光を入射し金薄膜
（２）側に生じるエバネッセンス波と表面プラズモンと
の共鳴する角度を測定して検出することを特徴とする表
面プラズモン共鳴の測定方法。6. A sensor chip generated by an electrochemical oxidation-reduction action by applying a predetermined potential to each electrode using the surface plasmon resonance enzyme sensor according to claim 5.
Changes in the electronic state, molecular aggregation state, chemical bonding state, etc. of the substance on the surface can be determined by the angle of resonance between the surface plasmon and the evanescent wave generated on the side of the gold thin film (2) when light enters the prism (7). A method for measuring surface plasmon resonance, characterized by measuring and detecting.
られた金または銀の金属薄膜と、この金属薄膜に設けら
れた該金属薄膜及び酵素の両方と電子移動反応を起こす
膜とを有するセンシング部分を含むことを特徴とする表
面プラズモン共鳴酵素センサー。7. An optically transparent substrate, a metal thin film of gold or silver provided on the substrate, and a film provided on the metal thin film and causing an electron transfer reaction with both the metal thin film and an enzyme. A surface plasmon resonance enzyme sensor comprising a sensing portion having:
ることを特徴とする請求項７に記載の表面プラズモン共
鳴酵素センサー。8. The surface plasmon resonance enzyme sensor according to claim 7, wherein the film causing the electron transfer is a polymer film.
介して前記金属薄膜と電荷のやり取りをする酵素を含む
ことを特徴とする請求項７に記載の表面プラズモン共鳴
酵素センサー。9. The surface plasmon resonance enzyme sensor according to claim 7, wherein the film that causes the electron transfer reaction includes an enzyme that exchanges electric charge with the metal thin film via the film.
該膜中に含まれる酵素を介して、またはその両方の中か
ら選択される１つと酸化還元反応を起こす分子を生成す
る性質を有する、酵素、細胞、微生物からなる群から選
択される１つを含むことを特徴とする請求項７に記載の
表面プラズモン共鳴酵素センサー。10. A film directly causing the electron transfer reaction,
One selected from the group consisting of enzymes, cells, and microorganisms, which has a property of generating a molecule that undergoes a redox reaction with one selected from among the enzymes contained in the membrane or both. The surface plasmon resonance enzyme sensor according to claim 7, comprising:
（１）の一方の面側に設けられた金薄膜（２）と、該金
薄膜（２）上に設けられた作用極（３ａ）、酸化還元高
分子膜（４）及び酵素固定膜（５）とを含むセンシング
部分（６）と、前記ガラス基板（１）の他方の面側に設
けられたプリズム（７）と、前記金薄膜（２）に接触し
て設けられ、前記作用極（３ａ）部分となる穴（８ａ）
を有するパッキン（８）を介して電解液を保持する空間
を有する治具（９）とを有する電気化学セル（１０）
と；該電気化学セル（１０）内に保持される電解液に接
して離間配置された対極（１１）および参照極（１２）
とを含み；これらの作用極（３ａ）、対極（１１）およ
び参照極（１２）をポテンシオスタットに接続して電気
化学的測定と表面プラズモン共鳴測定とを同時に行うよ
うに構成されたことを特徴とする表面プラズモン共鳴酵
素センサー。11. A glass substrate (1), a gold thin film (2) provided on one side of the glass substrate (1), and a working electrode (3a) provided on the gold thin film (2). Sensing part (6) including a redox polymer film (4) and an enzyme immobilized film (5), a prism (7) provided on the other surface side of the glass substrate (1), and the gold thin film A hole (8a) provided in contact with (2) and serving as the working electrode (3a) part;
(10) comprising a jig (9) having a space for holding an electrolyte through a packing (8) having
And a counter electrode (11) and a reference electrode (12) spaced apart from and in contact with the electrolyte held in the electrochemical cell (10).
That the working electrode (3a), the counter electrode (11) and the reference electrode (12) are connected to a potentiostat to perform electrochemical measurement and surface plasmon resonance measurement at the same time. Features a surface plasmon resonance enzyme sensor.
酵素センサーを用い、各電極に所定の電位を印加し、電
気化学的酸化還元作用によって生じるセンシング部分
（６）表面上の物質の電子状態、分子の集合状態、化学
的結合状態および物質移動からなる少なくとも１つの変
化を、プリズム（７）に光を入射し金薄膜（２）側に生
じるエバネッセンス波と表面プラズモンとの共鳴する角
度を測定して検出する工程を含むことを特徴とする表面
プラズモン共鳴の測定方法。12. Using the surface plasmon resonance enzyme sensor according to claim 11, a predetermined potential is applied to each electrode, and an electronic state and a molecule of a substance on the surface of the sensing portion (6) generated by an electrochemical redox action. At least one change consisting of the aggregation state, the chemical bonding state, and the mass transfer is measured by measuring the angle of resonance between an evanescent wave generated on the gold thin film (2) side and the surface plasmon when light is incident on the prism (7). A method for measuring surface plasmon resonance, comprising a step of detecting.
とも一部に設けられた表面プラズモン共鳴を起こし得る
物質を含む薄膜とを有するセンシング部を含み、該薄膜
は、該薄膜または該薄膜上に設けられた導体と電気化学
反応する物質と、酵素との両方により修飾されているこ
とを特徴とする表面プラズモン共鳴酵素センサー。13. A sensing unit having an optically transparent body and a thin film provided on at least a part of the transparent body and containing a substance capable of causing surface plasmon resonance, wherein the thin film is the thin film or the thin film A surface plasmon resonance enzyme sensor characterized by being modified by both an enzyme and a substance that electrochemically reacts with a conductor provided thereon.
する請求項１３に記載の表面プラズモン共鳴酵素センサ
ー。14. The surface plasmon resonance enzyme sensor according to claim 13, wherein the thin film is a polymer.
求項１３に記載の表面プラズモン共鳴酵素センサー。15. The surface plasmon resonance enzyme sensor according to claim 13, wherein the thin film is a gold or silver thin film.
り取りをする酵素を含むことを特徴とする請求項１３に
記載の表面プラズモン共鳴酵素センサー。16. The surface plasmon resonance enzyme sensor according to claim 13, wherein the thin film contains an enzyme that exchanges electric charges through the thin film.
素を介して、またはその両方の中から選択される１つと
酸化還元反応を起こす分子を生成する性質を有する、酵
素、細胞および微生物からなる群から選択される１つを
有することを特徴とする請求項１３に記載の表面プラズ
モン共鳴酵素センサー。17. An enzyme, cell, or microorganism having a property of generating a molecule that causes an oxidation-reduction reaction with one selected from the thin film directly, via an enzyme contained in the film, or both. The surface plasmon resonance enzyme sensor according to claim 13, comprising one selected from the group consisting of:
（１）の一方の面側に設けられた金薄膜（２）と、該金
薄膜（２）上に設けられた作用極（３ａ）、酸化還元高
分子膜（４）及び酵素固定膜（５）とを含むセンシング
部分（６）と、前記金薄膜（２）に接触して設けられ、
前記作用極（３ａ）部分となる穴（８ａ）を有するパッ
キン（８）を介して電解液を保持する空間を有する治具
（９）とを有する電気化学セル（１０）と；該電気化学
セル（１０）内に保持される電解液に接して離間配置さ
れた対極（１１）および参照極（１２）とを含み；これ
らの作用極（３ａ）、対極（１１）および参照極（１
２）を用いて電気化学的測定と表面プラズモン共鳴測定
とを同時に行うように構成されたことを特徴とする表面
プラズモン共鳴酸素センサー。18. A glass substrate (1), a gold thin film (2) provided on one side of the glass substrate (1), and a working electrode (3a) provided on the gold thin film (2). A sensing part (6) including a redox polymer membrane (4) and an enzyme-immobilized membrane (5); and a gold thin film (2).
An electrochemical cell (10) having a jig (9) having a space for holding an electrolyte through a packing (8) having a hole (8a) serving as the working electrode (3a); and the electrochemical cell. (10) a counter electrode (11) and a reference electrode (12) spaced apart from and in contact with the electrolytic solution held in the working electrode (3a), the counter electrode (11) and the reference electrode (1).
A surface plasmon resonance oxygen sensor characterized in that the electrochemical measurement and the surface plasmon resonance measurement are simultaneously performed by using 2).
酵素センサーを用い、各電極に酸化還元高分子膜（４）
または酵素固定膜（５）に電気化学的反応を起こさせる
電位を印加、または時間的に変化する電位を印加し、電
気化学的酸化還元作用によって生じるセンシング部分
（６）の物質の電子状態、分子の集合状態、化学的結合
状態及び物質移動からなる群から選択される少なくとも
１つの変化を、金薄膜（２）と反対側のガラス基体
（１）側から光を入射し、金薄膜（２）で起こる表面プ
ラズモン共鳴現象に基づいて検出する工程を含むことを
特徴とする表面プラズモン共鳴の測定方法。19. A redox polymer film (4) on each electrode using the surface plasmon resonance enzyme sensor according to claim 18.
Alternatively, a potential that causes an electrochemical reaction or a time-varying potential is applied to the enzyme-immobilized membrane (5), and the electronic state and molecule of the substance of the sensing portion (6) generated by the electrochemical redox action are applied. At least one change selected from the group consisting of an aggregation state, a chemical bonding state, and a mass transfer of light from the glass substrate (1) side opposite to the gold thin film (2); A method for measuring surface plasmon resonance, comprising a step of detecting based on a surface plasmon resonance phenomenon occurring in step (a).
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